Regioselective ortho-Hydroxylations of Flavonoids by Yeast

Article abstract of DOI:10.1021/acs.jafc.6b02210

Natural flavonoids, such as naringenin, hesperetin, chrysin, apigenin, luteolin, quercetin, epicatechin, and biochanin A, were subjected to microbiological transformations by Rhodotorula glutinis. Yeast was able to regioselectively C-8 hydroxylate hesperetin, luteolin, and chrysin. Naringenin was transformed to 8- and 6-hydroxyderivatives. Quercetin, epicatechin, and biochanin A did not undergo biotransformation. A metabolic pathway for the degradation of chrysin has been elucidated. The metabolism of chrysin proceeds via an initial C-8 hydroxylation to norwogonin, followed by A-ring cleavage to 4-hydroxy-6-phenyl-2H-pyran-2-one.

Full text of DOI:10.1021/acs.jafc.6b02210

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Article
Regioselective ortho-hydroxylations of flavonoids by yeast
Sandra Sordon, Anna Madej, Jaros#aw Pop#o#ski, Agnieszka Bartma#ska,
Tomasz Tronina, Ewa Brzezowska, Piotr Juszczyk, and Ewa Huszcza
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02210 • Publication Date (Web): 21 Jun 2016
Downloaded from http://pubs.acs.org on June 23, 2016
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Regioselective ortho-hydroxylations of flavonoids by yeast
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Sandra Sordon *, Anna Madej , Jarosław Popłoński , Agnieszka Bartmańska , Tomasz
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Tronina , Ewa Brzezowska , Piotr Juszczyk , Ewa Huszcza
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Department of Chemistry, Wrocław University of Environmental and Life Sciences,
Norwida 25, 50ꢀ375 Wroclaw, Poland
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Department of Biotechnology and Food Microbiology, Wrocław University of
Environmental and Life Sciences, Chełmońskiego 37/41, 51ꢀ630 Wrocław, Poland
*) Corresponding author: Sandra Sordon
Tel.: 0048713205197, Fax: 00483207744, Eꢀmail addresses: sandra.sordon@up.wroc.pl
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ABSTRACT
Natural flavonoids, such as naringenin, hesperetin, chrysin, apigenin, luteolin,
quercetin, epicatechin and biochanin A were subjected to microbiological transformations by
Rhodotorula glutinis. Yeast was able to regioselective Cꢀ8 hydroxylation of hesperetin, luteolin
and chrysin. Naringenin was transformed to 8ꢀ and 6ꢀhydroxyderivatives. Quercetin,
epicatechin and biochanin A did not undergo biotransformation. A metabolic pathway for the
degradation of chrysin has been elucidated. The metabolism of chrysin proceeds via an initial
Cꢀ8 hydroxylation to norwogonin, followed by Aꢀring cleavage to 4ꢀhydroxyꢀ6ꢀphenylꢀ2Hꢀ
pyranꢀ2ꢀone.
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Keywords: flavonoids, biotransformation, yeast, Rhodotorula glutinis, hydroxylation,
regioselectivity
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INTRODUCTION
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Flavonoids are a diverse group of ubiquitous plant secondary metabolites. Health
benefits associated with them resulted in a significant increase of interest in this class of
compounds. Some flavonoids, such as naringenin, naringin, hesperidin or chrysin, are
inexpensively available in a large quantities (see supplementary data). They may serve as a
viable source of lead compounds for the development of food additives, preservatives,
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antioxidants and also drug components.
Biotransformation is one of the possible methods for the preparation of various
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, 3
derivatives of flavonoids. The use of microorganisms as a biocatalyst is a relevant strategy to
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obtain high added value natural compounds from both cheap and readily available substrates.
These processes offer more advantages compared to conventional catalysts such as mild
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, 5
reaction conditions, high catalytic activities with high regioꢀ and stereoselectivity. Moreover,
according to the European Union Law, the products obtained by biotransformation of natural
compounds may be considered as natural ones (EU Directive 88/388/EEC).
It has been reported that that a protective effect of diet flavonoids against several
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diseases, among them atherosclerosis , is connected to their antioxidant properties. It is general
considered that a higher number of hydroxyl groups in a flavonoid results in a higher
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antioxidant activity. Briefly, the presence of two hydroxyl groups in an ortho position of ring
B, especially the 3´,4´ꢀdihydroxyphenolic structure, is the most important structure for
scavenging actions. Also of importance is the presence of hydroxyl groups at positions 5, 6 and
7 in the ring A. The ability of flavonoids to chelate metals results from the presence metal
complexing domains between the 5ꢀhydroxyl and 4ꢀcarbonyl group, the 3ꢀhydroxyl and 4ꢀ
carbonyl group and between the 3´,4´ꢀhydroxyl groups. Consequently, development of
microbial hydroxylation of flavonoids is significant in the synthesis of active antioxidants for
biological activity in human.
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Our previous microbial transformation research indicated the capability of the yeast
Rhodotorula marina to efficient hydroxylation of naringenin at Cꢀ6 or Cꢀ8 position to
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carthamidin and isocarthamidin, respectively.
These metabolites showed significantly
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greater antioxidant activity than either naringenin and its glycoside naringin. These
encouraging results prompted us to search for new useful red yeast biocatalysts, and then to
undertake studies on preparation of valuable derivatives of natural flavonoids by
biotransformation in the selected yeast cultures.
MATERIALS AND METHODS
Compounds
Naringenin (1), hesperidin, chrysin (3), biochanin A, (ꢀ)ꢀepicatechin, quercetin, ascorbic acid
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and DMSOꢀd were purchased from SigmaꢀAldrich, Sephadex LHꢀ20 from Pharmacia,
acetonitrile HPLC grade, silica gel 60 and TLC plates from Merck, peptone from Biocorp
(Poland). Other chemicals and solvents were purchased from POCH (Poland).
Hesperetin (2) was obtained by acidic hydrolysis of hesperidin.
A solution of 1.25 g of hesperetin (2) (≥80%), 500 mg of ascorbic (added to prevent oxidation
of the products of hydrolysis), 50 mL of 2M hydrochloric acid and 50 mL of methanol was
2 3
heated under reflux for 10 h. After cooling, Na CO was added to reach the pH about 4 and the
reaction mixture was extracted three times with 50 mL of ethyl acetate. The combined fractions
were washed with distilled water, dried over anhydrous magnesium sulfate and concentrated.
After purification by column chromatography on silica gel 60 using mixture chloroform :
methanol (10 : 1, v/v) as eluent, 307.0 mg of hesperetin (2) was obtained with purity greater
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than 95% (according to HPLC). H NMR (600 MHz, DMSOꢀd
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) δ (ppm): 2.70 (dd, 1H, J =
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17.1, 3.1 Hz, Hꢀ3ax), 3.18 (dd, 1H, J = 17.1, 12.3 Hz, Hꢀ3eq), 3.77 (s, 3H, C4´ꢀOꢀCH ), 5.42
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(dd, 1H, J = 12.3, 3.1 Hz, Hꢀ2), 5.88 (d, 1H, J = 2.1 Hz, Hꢀ6), 5.89 (d,1H, J = 2.1 Hz, Hꢀ8),
6.87 (dd, 1H, J = 8.3, 2.1 Hz, Hꢀ6´), 6.92 (d, 1H, J = 2.0 Hz, Hꢀ2´), 6.93 (d, 1H, J = 8.3 Hz, Hꢀ
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5´), 8.29 (s, 1H, 3´ꢀOH), 9.12 (s, 1H, 7ꢀOH), 12.12 (s, 1H, 5ꢀOH). C NMR (150 MHz,
DMSOꢀd ) δ (ppm): 42.1 (Cꢀ3), 55.8 (C4´ꢀOꢀCH ), 78.3 (Cꢀ2), 95.1 (Cꢀ8), 95.9 (Cꢀ6), 101.9
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(Cꢀ10), 112.1 (Cꢀ5´), 114.1 (Cꢀ2´), 117.8 (Cꢀ6´), 131.2 (Cꢀ1´), 146.5 (Cꢀ3´), 148.0 (Cꢀ4´),
162.9 (Cꢀ9), 163.6 (Cꢀ5), 166.7 (Cꢀ7), 196.3 (Cꢀ4).
Apigenin (4) was obtined from naringenin (1) by 2,3ꢀdichloroꢀ5,6ꢀdicyanoꢀ1,4ꢀbenzoquinone
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(DDQ) ꢀ oxidation of C2ꢀC3 bond according to the method described by Kim et al. H NMR
(600 MHz, DMSOꢀd ) δ (ppm): 6.19 (m, 1H, Hꢀ8), 6.48 (m, 1H, Hꢀ6), 6.77 (s, 1H, Hꢀ3), 6.92
(m, 2H, Hꢀ3´,5´), 7.92 (m, 2H, Hꢀ2´, 6´), 10.35 (s, 1H, 4´ꢀOH), 10.82 (s, 1H, 7ꢀOH), 12.96 (s,
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1H, 5ꢀOH). C NMR (150 MHz, DMSOꢀd ) δ (ppm): 94.0 (Cꢀ6), 98.8 (Cꢀ8), 102.9 (Cꢀ3),
103.7 (Cꢀ10), 116.0 (Cꢀ3´,5´), 121.2 (Cꢀ1´), 128.5 (Cꢀ2´,6´), 157.3 (Cꢀ5), 161.2 (Cꢀ4´), 161.5
(Cꢀ9), 163.7 (Cꢀ2), 164.1 (Cꢀ7), 181.8 (Cꢀ4).
Luteolin (5) was isolated from artichoke (Cynara scolymus L.) leaf dry extract. Flavonoid
glycosides included in the dry extract of artichoke were hydrolyzed. For this purpose a mixture
of 9 g of the extract, 200 mg of ascorbic (added to prevent oxidation of the products of
hydrolysis), 240 mL of 2M hydrochloric acid and 240 mL of methanol was heated under reflux
for 6 h. After cooling, Na CO was added to reach the pH about 4 and the reaction mixture was
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extracted three times with 250 mL of ethyl acetate. The combined fractions were washed with
distilled water, dried over anhydrous magnesium sulfate and concentrated to give 2.75 g of
extract. After purification by column chromatography on Sephadex LHꢀ20 using methanol as
eluent, 86.2 mg of luteolin (5) was obtained with purity greater than 98% (according to HPLC).
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H NMR (600 MHz, DMSOꢀd ) δ (ppm): 6.18 (d, 1H, J = 1.80 Hz, Hꢀ8), 6.44 (d, 1H, J = 1.8
Hz, Hꢀ6), 6.68 (s, 1H, Hꢀ3), 6.88 (d, 1H, J = 8.2 Hz, Hꢀ5´), 7.39 (s, 1H, Hꢀ2´), 7.41 (d, 1H, J =
9.6 Hz, 6´ꢀH), 9.49 (s, 1H, 4´ꢀOH), 9.99 (s, 1H, 3´ꢀOH), 10.84 (s, 1H, 7ꢀOH), 12.98 (s, 1H, 5ꢀ
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OH). C NMR (150 MHz, DMSOꢀd ) δ (ppm): 93.9 (Cꢀ8), 98.9 (Cꢀ6), 102.9 (Cꢀ3), 103.8 (Cꢀ
10), 113.4 (Cꢀ2´), 116.1 (Cꢀ5´), 119.1 (Cꢀ6´), 121.5 (Cꢀ1´), 145.8 (Cꢀ3´), 149.8 (Cꢀ4´), 157.4
(Cꢀ9), 161.5 (Cꢀ5), 164.0 (Cꢀ2), 164.2 (Cꢀ7), 181.7 (Cꢀ4).
Analytic procedures
TLC was carried out on silica gel 60 F254 (0.2 mm thick) plates. Compounds were detected by
4 2 3 3 10 4 2 4
spraying the plates with 1% Ce(SO ) and 2% H [P(Mo O ) ] in 10% H SO . HPLC was
performed on a Waters 2695 Alliance instrument with a photodiode array detector Waters 2996
(detection at 290 and 370 nm wavelength) equipped with the analytical HPLC column Agilent
ZORBAX Eclipse XDB 5 ꢁm (46 × 250 mm), which was eluted at 0.8 mL min. A linear
gradient was composed of (A) acetonitrile and (B) 0.1% acetic acid in water. After injection
solvent A increased from 25% to 38% over 10 min and held at 38% for 18 min, then further
decreased from 38% to 25% over 2 min, and finally held 25% for 6 min.
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NMR spectra ( H NMR, C NMR, DEPT 135°, Hꢀ H NMR (COSY), and Hꢀ C NMR
(HSQC, HMBC)) were recorded on a MHz DRX Bruker Avance™ 600 (600 MHz) instrument
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in DMSOꢀd . OD 600 nm analysis were performed using a Spectrofotometer Cintra 303, GBC,
Positiveꢀion HRꢀESIMS spectra were measured on a Bruker micrOTOFꢀQ spectrometer.
Microorganisms
Microorganisms used in this work were purchased from Institute of Biology and Botany,
Wrocław Medical University (indexed AM) and Department of Chemistry, Wrocław
University of Environmental and Life Sciences (indexed KCh). The red yeast cultures used for
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preliminary study were as follows: Rhodotorula glutinis AM242, Rhodotorula glutinis
KCh735, Rhodotorula rubra AM4 and Rhodotorula rubra AM82.
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Biotransformation procedure
The yeast were cultivated on rotary shakers (130 rpm, 6.5 amplitude) at 28 °C in 100ꢀ
mL Erlenmeyer flasks with 30 mL of the medium in the preliminary screening experiments and
in 300ꢀmL Erlenmeyer flasks containing 100 mL of the medium in the preparative scale
fermentation. The growth medium for biotransformation processes (Sabouraud medium)
contained 3% glucose and 1% peptone. The cultures on agar slants were used to obtain the
inoculation culture, and then 2ꢀday inoculation culture were transferred to the main cultures
media (1 mL to the 30 mL and 3 mL to the 100 mL). After three days of growth the substrates
were added.
In the screening studies, 5 mg of naringenin (1) dissolved in 0.5 mL of acetone was
added to the culture. Experiments were performed in triplicate. Biotransformations were
performed for 48 hours, acidified using 1M HCl to pH about 5. Then each of the reaction
mixture was extracted with ethyl acetate (2 x 10 mL).
In the preparative biotransformations, a total of 80 mg of each substrate dissolved in 4 mL of
methanol (for substrates 1, 2, 5) and DMSO (for substrates 3, 4) or was equally distributed
among four flasks with 3ꢀday fungal cultures of OD600 = 22.40 ± 0.99 (1 mL each) to give a
final concentration of 200 mg / L. Reactions were carried out for 1 to 6 days, acidified using
1M HCl to pH about 5, and then each of the reaction mixture was extracted with ethyl acetate
(3 x 40 mL).
The progress of biotransformations was monitored by TLC and HPLC. Substrate
controls consisted of substrate and a sterile growth medium incubated without microorganism.
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Preparative transformation of chrysin (3) leading to its degradation was carried out
under similar conditions except that tetrahydrofuran was used to dissolve the substrate.
Products isolation and analysis
Extracts were dried with anhydrous magnesium sulphate, filtered and evaporated to
dryness. The resulting materials were redissolved in methanol and analyzed by TLC and
HPLC. The combined organic phases were evaporated and column chromatographed over
Sephadex LHꢀ20 gel using chloroform/methanol (4:1 v/v) as eluent for 1, 3, 4 and 5, and
chloroform/methanol (10:1 v/v) for 2. Quantitative analysis of the ethyl acetate extract of the
biotransformation mixture was performed by means of HPLC, using calibration curves for the
substrate and for all the isolated products.
The structures of the obtained compounds were confirmed by NMR spectra analysis.
10, 11, 13ꢀ19
Spectroscopic data of the products 6ꢀ12 are with agreement with literature data.
carthamidin (5, 6, 7, 4´ꢀ tetrahydroxyflavanone, 6)
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H NMR (600 MHz, DMSOꢀd ) δ (ppm): 2.65 (dd, 1H, J = 17.1, 2.9 Hz, Hꢀ3eq), 3.22 (dd, 1H,
J = 17.1, 12.5 Hz, Hꢀ3ax), 5.36 (dd, 1H, J = 12.5, 2.9 Hz, Hꢀ2), 5.94 (s, 1H, Hꢀ8), 6.79 (d, 2H, J
= 8.5 Hz, Hꢀ3´, 5´), 7.31 (d, 2H, J = 8.5 Hz, Hꢀ2´,6´), 8.11 (s, 1H, 7ꢀOH), 9.57 (s, 1H, 4´ꢀOH),
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10.42 (s, 1H, 6ꢀOH), 11.98 (s, 1H, 5ꢀOH). C NMR (150 MHz, DMSOꢀd ) δ (ppm): 42.3 (Cꢀ
3), 78.5 (Cꢀ2), 94.7 (Cꢀ8), 101.7 (Cꢀ10), 115.2 (Cꢀ3´, 5´), 126.3 (Cꢀ6), 128.3 (Cꢀ2´, 6´), 129.2
(Cꢀ1´), 150.2 (Cꢀ9), 155.3 (Cꢀ5), 155.8 (Cꢀ7), 157.7 (Cꢀ4´), 196.7 (Cꢀ4).
isocarthamidin (5, 7, 8, 4´ꢀ tetrahydroxyflavanone, 7)
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H NMR (600 MHz, DMSOꢀd ) δ (ppm): 2.71 (dd, 1H, J = 17.1, 3.0 Hz, Hꢀ3eq), 3.22 (dd, 1H,
J = 17.1, 12.3 Hz, Hꢀ3ax), 5.42 (dd, 1H, J = 12.3, 3.0 Hz, Hꢀ2), 5.93 (s, 1H, Hꢀ6), 6.79 (d, 2H, J
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= 8.5 Hz, Hꢀ3´, 5´), 7.35 (d, 2H, J = 8.5 Hz, Hꢀ2´, 6´), 8.11 (s, 1H, 7ꢀOH), 9.57 (s, 1H, 4´ꢀOH);
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10.42 (s, 1H, 8ꢀOH), 11.75 (s, 1H, 5ꢀOH). C NMR (150 MHz, DMSOꢀd ) δ (ppm): 42.3 (Cꢀ
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3), 78.5 (Cꢀ2), 95.4 (Cꢀ6), 101.7 (Cꢀ10), 115.1 (Cꢀ3´,5´), 125.6 (Cꢀ8), 128.4 (Cꢀ2´, 6´), 129.2
(Cꢀ1´), 149.4 (Cꢀ9), 156.5 (Cꢀ5), 155.8 (Cꢀ7), 157.7 (Cꢀ4´), 196.6 (Cꢀ4).
8ꢀhydroxyhesperetin (3´,5,7,8ꢀtetrahydroxyꢀ4´ꢀmetoxyflavanone, 8)
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H NMR (600 MHz, DMSOꢀd ) δ (ppm): 2.72 (1H, dd, J = 17.1, 3.1 Hz, Hꢀ3eq); 3.15 (1H, dd,
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J = 11.8 Hz, 17.1 Hz, Hꢀ3ax), 3.77 (s, OCH ), 5.41 (1H, dd, J = 11.8, 3.1 Hz, Hꢀ2), 5.92 (1H, s,
Hꢀ6), 6.91 (1H, d, J = 1.8 Hz, Hꢀ2´), 6.94 (1H, dd, J =12.9, 1.8 Hz, Hꢀ6´), 6.95 (1H, d, J = 12.1
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Hz, Hꢀ5´). C NMR (150 MHz, DMSOꢀd ) δ (ppm): 42.4 (Cꢀ3), 55.8 (OCH ), 78.3 (Cꢀ2), 95.5
(Cꢀ6), 101.8 (Cꢀ10), 112.0 (Cꢀ2´), 114.3 (Cꢀ5´), 117.8 (Cꢀ6´), 125.7 (Cꢀ8), 131.5 (Cꢀ1´), 146.5
(Cꢀ3´), 147.9 (Cꢀ4´), 149.3 (Cꢀ9), 155.8 (Cꢀ7), 156.5 (Cꢀ5), 196.5 (Cꢀ4).
norwogonin (5,7,8ꢀtrihydroxyflavone, 9)
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H NMR (600 MHz, DMSOꢀd ) δ: 6.30 (1H, s, Hꢀ6), 6.94 (1H, s, Hꢀ3), 7.59 (3H, m, Hꢀ3´, 4´,
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5´), 8.16 (2H, m, Hꢀ2´,6´), 12.26 (1H, s, 5ꢀOH). C NMR (150 MHz, DMSOꢀd ) δ (ppm): 98.8
(Cꢀ6), 103.6 (Cꢀ10), 104.7 (Cꢀ3), 125.2 (Cꢀ8), 126.6 (Cꢀ2´, 6´), 129.1 (Cꢀ3´, 5´), 130.9 (Cꢀ1´),
132.0 (Cꢀ4´), 145.6 (Cꢀ9), 153.1 (Cꢀ5), 153.8 (Cꢀ7), 163.0 (Cꢀ2), 182.3 (Cꢀ4).
4ꢀhydroxyꢀ6ꢀphenylꢀ2Hꢀpyranꢀ2ꢀone (10)
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H NMR (600 MHz, DMSOꢀd ) δ: 5.40 (1H, m, Hꢀ3), 6.75 (1H, m, Hꢀ5), 7.49ꢀ7.54 (3H, m, Hꢀ
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3´, 4´, 5´), 7.81ꢀ7.87 (2H, m, Hꢀ 2´, 6´). C NMR (150 MHz, DMSOꢀd ) δ (ppm): 89.7 (Cꢀ3),
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98.5 (Cꢀ5) 125.6 (Cꢀ2´, 6´), 129.3 (Cꢀ3´, 5´), 131.1 (Cꢀ1´), 131.2 (Cꢀ4´), 160.25 (Cꢀ4), 163.5
+
(Cꢀ6), 170.8 (Cꢀ2). HRESIꢀMS [M+H] (calculated/found) (m/z 189.0546/189.0545).
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8ꢀhydroxyapigenin (4´,5,7,8ꢀtetrahydroxyflavone, 11)
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H NMR (600 MHz, DMSOꢀd ) δ: 6.26 (1H, s, Hꢀ6), 6.74 (1H, s, Hꢀ3), 6.93 (2H, m, Hꢀ3´, 5´),
6
8.01 (2H, m, Hꢀ2´,6´), 8.75 (1H, s, 8ꢀOH), 10.33 (1H, s, 4´ꢀOH), 10.46 (1H, s, 7ꢀOH), 12.38
13
6
(1H, s, 5ꢀOH). C NMR (150 MHz, DMSOꢀd ) δ (ppm): 98.6 (Cꢀ6), 102.3 (Cꢀ3), 103.3 (Cꢀ
10), 115.9 (Cꢀ3´, 5´), 121.4 (Cꢀ1´), 125.0 (Cꢀ8), 128.6 (Cꢀ2´, 6´), 145.5 (Cꢀ9), 153.0 (Cꢀ5),
153.4 (Cꢀ7), 161.1 (Cꢀ4´), 163.5 (Cꢀ2), 182.1 (Cꢀ4).
hypoletin (3´,4´,5,7,8ꢀpentahydroxyflavone, 12)
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H NMR (600 MHz, DMSOꢀd ) δ (ppm): 6.26 (1H, s, Hꢀ6), 6.63 (1H, s, Hꢀ3), 6.89 (1H, d, J =
13
6
8.2 Hz, Hꢀ5´), 7.49 (2H, d, J = 12.6 Hz, Hꢀ2´, 6´); C NMR (150 MHz, DMSOꢀd ) δ (ppm):
98.6 (Cꢀ6), 102.4 (Cꢀ3), 103.4 (Cꢀ10), 113.7 (Cꢀ 2´), 116.0 (Cꢀ5´), 119.2 (Cꢀ6´), 121.8 (Cꢀ1´),
125.1 (Cꢀ8), 145.6 (Cꢀ3´), 145.7 (Cꢀ5), 149.7 (Cꢀ4´), 153.0 (Cꢀ7), 153.3 (Cꢀ9), 163.8 (Cꢀ2),
182.1 (Cꢀ4).
RESULTS AND DISCUSSION
Screening test carried out on four red yeasts cultured on Sabouraud medium with
naringenin (1) as a model substrate lead to selection of Rhodotorula glutinis KCh735 strain,
which was capable for transformation of 1. The oleaginous species of Rhodotorula glutinis can
be potentially used for biodiesel production and also other important products, such as
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carotenoids.
Our studies carried out on a larger scale with the use of four natural flavonoids, having
flavanone (1, 2), flavone (3, 4, 5), flavonol (quercetin), flavane (epicatechin) and isoflavone
(biochanin A) skeletons, have demonstrated the ability of this organism to perform
hydroxylation reaction of the first two flavonoid groups (Fig. 1). Hydroxylation reaction is the
capacity rarely observed in yeast. Preparativeꢀscale biotransformations afforded seven
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metabolites (6ꢀ12). The structures of the products were identified and confirmed by comparison
10, 11, 13ꢀ19
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of the spectroscopic data with those reported in literature.
Naringenin (1) after 24 h was converted to carthamidin (6) and isocarthamidin (7) in a
ratio of 1 : 19, respectively (according to HPLC). The starting material was not observed. The
35.8 mg of mixture of 5 and 6 was isolated with the yield of 42.3%. These metabolites were
investigated by Miyake et al. by using of free radicalꢀscavening system of 1,1ꢀdiphenylꢀ2ꢀ
11
picrylhydrazyl (DPPH) and the methyl linoleate oxidative system. In both of these assays
they exhibited higher antioxidant activity than the naringenin (1). The activity of the
isocarthamidin (7) was stronger that of carthamidin (6) and nearly equal to the αꢀtocopherol.
The both products of naringenin (1) biotransformation were also recognized and patented as a
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antidiabetic agents. Recent study on the screening of new tyrosinase inhibitors resulted in the
identification of isocarthamidin (5,7,8,4´ꢀtetrahydroxyflavanone, 7), and other ortoꢀ
hydroxylated flavonoids (6,7,4´ꢀtrihydroxyisoflavone, 7,8,4´ꢀtrihydroxyisoflavone and 5,7,8,4´ꢀ
23ꢀ25
tetrahydroxyisoflavone) as a potent tyrosinase inhibitors.
Cꢀ6 and Cꢀ7 hydroxyl groups in
the Aꢀring strongly affect both the inhibitory strength and the inhibitory mode of the flavonoids
on mushroom tyrosinase. Inhibiting the tyrosinase activity has been the subject of many studies
because this enzyme is responsible among others for the undesirable hyperpigmentation in skin
26
and browning in fruits. In light of these information the yeast Rhodotorula glutinis which lead
regioselective hydroxylation in these key positions flavonoids may be a very useful tool for
obtaining natural inhibitors of tyrosinase.
27
The content of 6 and 7 in plants is very low , therefore from a pharmaceutical point of view
the preparation of these valuable compounds in an efficient biotransformation of much cheaper
substrate (see supplementary data) is very promising. Biohydroxylation of 1 at position 6 or 8
11
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was earlier described for filamentous fungi Aspergillus saitoi , Aspergillus niger and for
9
,10
yeast Rhodotorula marina. In the process using the yeast R. marina substrate conversion and
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the ratio of products in the biotransformation mixture were comparable to results obtained for
the fungus A. niger (the maximum concentration of isocarthamidin was 21.7 mg/L and
carthamidin 21.5 mg/L, with the initial concentration naringenin equal to 60 mg/L), however 5ꢀ
fold more of the substrate can be introduced to the yeast culture (300 mg /L).
The results of biotransformations described in this article, concerning another species of
red yeast ꢀ Rhodotorula glutinis, indicate high similarity in metabolizing of 1 for both
organisms. The application of R. glutinis gave acceptable ratio of more valuable isocarthamidin
(7) to carthamidin (6) (1:19), but the most important advantage of the newly selected
biocatalyst is its ability to regioselective orthoꢀhydroxylation of other natural flavonoids. The
tested yeast was capable of converting flavonoids that belong to the flavanones and flavones
groups, but did not metabolize substrates having flavonol, flavane and isoflavone skeletons.
Biotransformations of hesperetin (2), chrysin (3), apigenin (4) and luteolin (5) are described
below.
The incubation of hesperetin (2) with Rhodotorula glutinis for 72 hours gave 8ꢀ
hydroxyhesperetin (8) (14.3 mg, 17.0%). The antioxidant activity of this compound
investigated by Miyake et al. is high, its ability to scavenge DPPH radicals is similar to αꢀ
11
tocopferol, at a concentration of 10 ꢁM was 63%.
8ꢀHydroxyhesperetin (8) was previously obtained by biotransformation of hesperetin
11
(2) in the cultures of filamentous fungi Aspergillus saitoi and Mucor ramannianus ATCC
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2628. Both of these transformations occurred with the low yield (3.5%, 0.03%) and ꢀ in the
case of Aspergillus saitoi ꢀ in a long process lasting 20 days. Our new method for preparing 8ꢀ
hydroksyhesperetin (8) by using the yeast Rhodotorula glutinis shortens the time of
transformation to three days and allows to obtain the product with the nearly five times higher
yield.
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Preparative scale 6ꢀday fermentation of chrysin (3) yielded one metabolite ꢀ
norwogonin (9) (26.2 mg, 30.8%), which is the one of the active components in the root of
Scutellaria baicalensis. According to literature, 9 is more effective than wogonin (5,7ꢀ
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dihydroxyꢀ8ꢀmethoxy flavone) in induction of apoptosis in leukemia HLꢀ60 cells. It is also
one of the most potent antimicrobial natural compound against multidrugꢀresistant bacteria
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Acinetobacter baumannii. Thus, the above described experiment may serve after optimizing
as a method for obtaining valuable norwogonin (9) from chrysin (3) ꢀ a cheap source, in a oneꢀ
step simple biotransformation process.
In the described biotransformation it was also observed the presence of trace amounts of
the second product, which interestingly was the main product in the analogous process, in
which chrysin (3) was added to the culture as a solution in tetrahydrofuran. Transformation of
chrysin (3) added to the yeast culture in tetrahydrofuran for 48 hours led to 4ꢀhydroxyꢀ6ꢀ
phenylꢀ2Hꢀpyranꢀ2ꢀone (10) (30.5 mg, 50.2%). The product had the molecular formula
+
C
11
H
8
O
3
as indicated by HRꢀESIMS of the [M+M] ion peak at m/z 189.0545 (calc. 189.0546).
13
The C NMR spectrum differed from that of starting compound (3) in the reduction of four
signals what suggested degradation of substrate. Signals of Bꢀring had nearly the same
chemical shifts as recorded for chrysin (3). Three signals at the lower field belongs to Cꢀring
carbons: Cꢀ2 (170.8 ppm), Cꢀ6 (163.5 ppm) and Cꢀ4 (160.3 ppm). Further evidence came from
1
the HꢀNMR spectrum which showed only four signals from seven protons. The absence of two
singlets at 12.8 ppm (C5ꢀOH), 10.92 ppm (C7ꢀOH) and two doublets at 6.22 ppm (Hꢀ6) and
6.52 ppm (Hꢀ8), presented in the spectrum of the substrate, confirmed degradation of the Aꢀ
ring of chrysin (3). Correlations observed in the HMBC spectrum permitted the assignment of
the Hꢀ3 and Hꢀ5 protons of lactone product (Fig. 2). The aromatic Aꢀring proton appeared as a
multiplet at 6.75 ppm resonated with four carbons: at 89.7 ppm (Cꢀ3), 131.1 ppm (Cꢀ1´), 160.3
ppm (Cꢀ4) and 170.8 ppm (Cꢀ2), what suggested that it is bonded to the carbon atom Cꢀ5. In
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turn the other Aꢀring proton appeared as a multiplet at 5.40 ppm resonated with three carbons:
at 98.5 ppm (Cꢀ5), 163.5 ppm (Cꢀ6) and 170.8 ppm (Cꢀ2). No correlation of this proton with
the Cꢀ1´ carbon atom indicates the considerable spatial distance between mentioned atoms and
unambiguously assigns it to the carbon Cꢀ3.
The chemical synthesis of 4ꢀhydroxyꢀ6ꢀphenylꢀ2Hꢀpyranꢀ2ꢀone (10) has been
18
described. No information is found on the direct use of the product 9, but it is considered that
compounds containing in their molecule the structure of 4ꢀhydroxyꢀ6ꢀphenylꢀ2Hꢀpyranꢀ2ꢀone
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can be HIV protease inhibitors so that they can show antiviral activity. The simple oneꢀpot
process for preparing 4ꢀhydroxyꢀ6ꢀphenylꢀ2Hꢀpyranꢀ2ꢀone (10) by biotransformation can be an
alternative to the mentioned three step chemical method.
Degradation of chrysin (3) in the culture of Rhodotorula glutinis, involving the breakꢀ
up of the Aꢀring, prompted us to investigate its course. The presence of the lactone moiety in
the product 10 allow to assume that an intermediate of this pathway have a hydroxyl group and
the Cꢀ8 position of flavone skeleton. This assumption is also consistent with the demonstrated
ability of yeast selected by us to orthoꢀhydroxylation of the A ring of other flavones and
flavanones. The OH groups makes the flavonoids capable of autooxidation under prolonged
aerobic environments, which becomes more rapid in alkaline conditions. The course of the Aꢀ
ring cleavage by Rhodotorula glutinis proceeds via an initial Cꢀ8 hydroxylation of chrysin (3)
to norwogonin (9), oxidative ringꢀfission followed by a hydrolaseꢀtype reaction to yield 4ꢀ
hydroxyꢀ6ꢀphenylꢀ2Hꢀpyranꢀ2ꢀone (10).
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Flavonoids were generally decomposed via Cꢀring fission.
To the best our
knowledge degradation of flavonoids via Aꢀring cleavage was described only for bacteria from
the genus Pseudomonas. Pseudomonas sp. grown on (+)ꢀcatechin, oxidized dihydrogossypetin
(3´,4´,5,7,8ꢀpentahydroxyflavanonol) by cleaving the Aꢀring to form 5ꢀ(3,4ꢀdihydroxyphenyl)ꢀ
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4ꢀhydroxyꢀ3ꢀoxovaleroꢀ8ꢀlactone and oxaloacetic acid. If the lactone ꢀ type intermediates
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have a Cꢀ3 hydroxyl group at an unsaturated site, they can be substrates for a second
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dioxygenase and underwent further degradation by Pseudomonas putida.
3ꢀ
Hydroxyflavanones, such as taxifolin, were not oxidized.
Chrysin (4) biotransformations described above indicate a substantial influence of the
solvent in which the substrate is administered, on the rate of its metabolism. Addition of
terahydrofuran to the well ꢀ grown yeast culture accelerates chrysin (4) degradation.
8ꢀHydroxyapigenin (11) was the only product of the 6ꢀday biotransformation of
apigenin (4). The amount of 10.9 mg of this compound was isolated with the yield of 12.9%.
Apigenin (4) is widely used as nutritional supplement. The rich source of 4 are among others
parsley and celery. 8ꢀHydroxyapigenin (11) significantly inhibited lung virus proliferation
38
when administered intranasally or orally to mice without being toxic against mice.
Biotransformation of luteolin (3) was carried out for 36 hours and hypoletin (12) was
the only product isolated (21.3 mg, 25.3%). Hypoletin (12) is present in plants in small
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quantities, and exhibits antiꢀinflammatory and antidiabetic activities.
To sum up, Rhodotorula glutinis KCh735 was found to be the only one of tested red
yeast strains, which showed the ability of naringenin biotransformation in the preliminary
studies. This selected strain is capable of regioselective orthoꢀhydroxylation of the Aꢀring of
flavonoids, that is unique in the phylum of yeasts. Described biotransformations of natural
flavones and flavanones using this biocatalyst are the method that allows to specifically convert
cheap natural bioactive molecules into rare and pharmacologically improved metabolites.
Further optimization of fermentation conditions may provide an efficient route for massive
production of this valuable substances.
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SUPPORTING INFORMATION
The Supporting Information is available free of charge on the ACS Publications website at
DOI:
Detailed spectroscopic and structural information of biotransformation substrates and products,
the approximate prices list of same used and obtained flavonoids (PDF).
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Funding Information
The Project was supported by Wroclaw Centre of Biotechnology, The Leading National
Research Centre Programme (KNOW) for years 2014ꢀ2018.
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FIGURE CAPTIONS
Figure 1. Transformation of naringenin (1), hesperetin (2), chrysin (3), apigenin (4) and
luteolin (5) by Rhodotorula glutinis.
Figure 2. Key HMBC correlations for compound 10.
Figure 3. Putative pathway of chrysin (3) degradation via Aꢀring cleavage conducted by
Rhodotorula glutinis.
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Figure 1
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Figure 2
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Figure 3
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